Limitin protein translated from the second ATG but not that from the first ATG has growth-inhibiting properties on BC7.12, a pre-B cell line.

(A) Constructs of the full cDNA/pEFBOS, 1st-ATG/pEFBOS, and 2nd-ATG/pEFBOS plasmids. The relative positions of the open reading frames of the limitin proteins with the translational start and stop codons are shown. The cDNA inserts of each plasmid are indicated by arrows. (B) The full cDNA/pEFBOS, 1st-ATG/pEFBOSX, or 2nd-ATG/pEFBOSX plasmids were transfected into 293T cells with a calcium phosphate precipitation method. The transfectants were cultured for 3 days, and then their supernatants were collected. BC7.12 cells (1 × 10⁴/well) were cultured in the presence of 10% of the indicated supernatants for 2 days. Their viable cell number was evaluated by a hemocytometer. Results are shown as means ± SDs of triplicate samples. Statistically significant differences from control values (P < .01) are indicated by **. Similar results were obtained in 3 independent experiments. (C) Whole cell lysates were prepared from 293T cells transfected with or without 1st-FLAG/CMV-5a, and subjected to Western blot analysis probed with anti-FLAG antibody. The arrow indicates the product from the 1st-FLAG/CMV-5a plasmid. (D) A fusion protein composed of the 33 amino acid short limitin protein and the FLAG tag was purified from cell lysates of 293T cells transfected with 1st-FLAG/CMV-5a by using anti-FLAG affinity chromatography. BC7.12 cells (1 × 10⁴/well) were cultured in the presence of the fusion protein or recombinant limitin for 2 days. Viable cell numbers were then evaluated with a hemocytometer. Results are shown as means ± SDs of triplicate samples. Statistically significant differences from control values (P < .01) are indicated by **.